This invention is in the field of molecular biology. In particular, this invention relates to the isolation, purification, cloning and expression of plant xyloglucan fucosyltransferases.
In most multicellular organisms, cells are embedded in an intricate extracellular matrix that keeps them together and influences the shape, development, and polarity of the cells they contact. Animal cells have such an extracellular matrix at their surface, but plants possess a distinct wall that encloses every cell. Many important differences between plants and animals with respect to nutrition, digestion, growth, reproduction, and defense mechanisms can be traced to the plant cell wall. Cell walls are mediators of growth, which in plants is determined largely by the wall extensibility provided that sufficient turgor pressure is present. Morphogenesis is also effected by the cell wall at the tissue and cellular levels. The biosynthesis of plant cell walls must be very tightly regulated. Although an individual plant cell may expand its volume by as much as 18,840 times, its cell wall must maintain a regular thickness and uniform structure to prevent hemorrhaging of the cell contents due to the high internal turgor pressure. However, despite extensive descriptions of the chemical and physical structure of the plant cell wall, very little is known about its biosynthesis. Only one cell wall-synthesizing glycosyltransferase, cellulose synthase, has been cloned and described in any detail
Plant cell walls are mainly composed of cellulose microfibrils and matrix polysaccharides. Hemicellulose is a type of matrix polysaccharide that binds tightly but noncovalently to cellulose microfibrils, helping to crosslink them into a complex network. Xyloglucan is a fundamentally important hemicellulose in dicot and nongraminaceous monocot plants. It comprises approximately 25% of the total cell wall and forms a load-bearing network by associating with the faces of surrounding cellulose microfibrils via hydrogen bonds. Xyloglucan contains a beta-1,4-glucan backbone decorated with side chains of xylose alone, xylose and galactose, and xylose, galactose and fucose. The presence or absence of the fucose residue is thought to determine whether the xyloglucan conformation is planar and thus better able to bind to cellulose, a critical step in cell wall formation. In addition, oligosaccharides consisting of a monomer of xyloglucan have been shown to prevent auxin-promoted elongation of pea stems when the oligosaccharides contain fucose, but not if they lack fucose suggesting that xyloglucan fragments act as signalling molecules in vivo. Xyloglucan fucosylation is thus a critical step in plant development.
There is thus a need to identify the genes and gene products involved in plant xyloglucan fucosylation. In particular, there is a need to isolate, purify and clone xyloglucan fucosyltransferase genes and gene products so that xyloglucan fucosylation may be controlled and regulated in plants and other organisms.
In order to meet these needs, the present invention is directed to purified, isolated, sequenced and cloned plant xyloglucan fucosyltransferase. In addition, the present invention is directed to the purification, isolation, sequencing and cloning of plant xyloglucan fucosyltransferase. The present invention is further directed to transgenic organisms expressing plant xyloglucan fucosyltransferase. The present invention is further directed to transgenic plants expressing regulated levels of xyloglucan fucosyltransferases.
In general, the invention features substantially pure fucosyltransferase DNA or protein obtained from a plant. In a related aspect, the invention features a fragment or analog polypeptide including an amino acid sequence substantially identical to the sequences shown in SEQ ID NOs: 1, 5 and 7.
In another related aspect the invention features substantially pure DNA having a sequence substantially identical to the nucleotide sequence shown in SEQ ID NOs: 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, and 15. In preferred embodiments, such DNA is cDNA or is genomic DNA. In related aspects, the invention also features a vector and a cell (e.g., a plant) which includes such substantially pure DNA. In various preferred embodiments, the vector-containing cell is a prokaryotic cell, for example, E. coli or Agrobacterium or, more preferably, a plant cell.
In yet another related aspect, the invention features a method of fucosylating a polypeptide in vivo involving: (a) providing a cell containing the fucosyltransferase DNA of the invention positioned for expression in the cell; and (b) culturing the transformed cell under conditions for expressing the DNA, resulting in the fucosylation of the protein. In preferred embodiments, fucosylation occurs in a plant cell.
In another aspect, the invention features a recombinant polypeptide fucosylated using a cell expressing DNA which is substantially identical to the nucleotide sequence shown in SEQ ID NOs: 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, and 15. In still other preferred embodiments, the polypeptide is further fucosylated using one or more fucosyltransferases.
The present invention further includes multiple types of DNA constructs including (1) xe2x80x9csensexe2x80x9d constructs encoding proteins, which can increase the expression of fucosyltransferases in plant species and (2) xe2x80x9cantisensexe2x80x9d constructs containing DNA, which can be used to produce antisense RNA in to reduce expression of fucosyltransferases in plants. Optimal amounts of antisense RNA in transgenic plants will selectively inhibit the expression of genes in these plants which are involved in the fucosylation of xyloglucans.
Some of these constructs will direct constitutive production of transcripts. Other constructs will direct expression in specific organs and/or specific tissue layers of the transgenic plant. These organs will include leaves, petioles, stems, flower organs, seeds, fruits or photosynthetically active parts of the plant. Tissue layers will include but may not be restricted to the epidermis and adjacent cell layers.
The present invention also provides recombinant cells and plants containing these constructs.
In one embodiment, the first category of DNA constructs include: a promoter selected from but not limited to constitutive, tissue-specific, cell-type specific, seed-specific, flower-specific, fruit-specific, epidermis-specific promoters, a promoter specific to cell layers adjacent to the epidermis or a promoter specific to photosynthetically active plant tissues, which functions in plant cells to cause the production of an RNA sequence. In this embodiment, the DNA coding region sequences that encode proteins which can be used to increase the activity of plant fucosyltransferases in transgenic plants. The DNA coding region will further include a region 3xe2x80x2 to the coding regions the 3xe2x80x2 non-translated region which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3xe2x80x2 end of the RNA sequence promoter.
In another embodiment, a second category of DNA construct will include a constitutive promoter, seed-specific, flower-specific, fruit-specific, epidermis-specific promoter, a promoter specific to cell layers adjacent to the epidermis or a promoter specific to photosynthetically active plant tissues, which functions in plant cells to cause the production of an RNA sequence. The DNA construct will also include DNA sequences which can produce antisense RNA molecules. These RNA molecules can selectively inhibit the accumulation of transcripts encoding proteins which encode plant fucosyltransferases.
In accordance with another aspect of the present invention, there is provided a method of producing genetically transformed plants which express a gene or genes involved in fucosyltransferase activity. In this method, a recombinant, double-stranded DNA molecule is incorporated into the genome of a plant cell. In this embodiment, the DNA sequence will include a promoter which functions in plant cells to cause the production of an RNA sequence in flowers, seeds, fruit or other plant tissues. In addition, the sequence will include a DNA coding sequence encoding proteins involved in fucosyltransferase activity in plants. Alternatively, the sequence will be a template to the synthesis of antisense RNA inhibiting the development of these structures. The DNA sequence will also include a 3xe2x80x2 non-translated region which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3xe2x80x2 end of the RNA sequences. The method also includes obtaining transformed plant cells and regenerating from the transformed plant cells genetically transformed plants. The transformed plant cells may be used to overproduce in cell culture the fucosylated xyloglucans.
The present invention is also directed to transgenic cells such as yeast, fungi, mammalian, and the like cells expressing the DNA sequences of this invention. The present invention is also directed to purified fucosylated xyloglucans isolated from the transgenic cells of the invention.